This R21 proposal is designed to test the hypothesis that one or more of the isoforms of carbonic anhydrase (CA) play an important role in both the etiology and treatment of insulin resistance. The data supporting a role for CA in insulin resistance comes from both the literature and our own preliminary data. CA enzymes catalyze the reaction CO2 + H2O >> H+ + HCO3-, and this HCO3- is required in two carboxylation reactions involved in the synthesis of malonyl-CoA. Malonyl-CoA plays a central role in cellular substrate utilization because it inhibits the activity of mitochondrial carnitine palmitoyl transferase-1 (CPT-1). Elevated malonyl-CoA thus reduces lipid oxidation and metabolism, and increases intracellular lipid accumulation. Recently, in muscle from obese subjects, it has been demonstrated that the accumulation of intracellular lipid, due to increased malonyl-CoA activity, produces insulin resistance, possibly via decreased insulin signaling. We now observe in muscle of obese humans and rodents that there are elevated levels of one isozyme, CA I (other isozymes have not yet been studied). Moreover, in cultured muscle cells, inhibition of CA isozymes increases insulin-mediated glucose transport. To provide additional data that CA isozymes are involved in both the etiology and treatment of insulin resistance, the proposed studies have three distinct Aims. First, we will test the effects of specific CA inhibitors on lipid oxidation and insulin-action in cultured muscle cells, to determine whether CA inhibition is a target for improving insulin sensitivity. Second, in muscle from obese humans, we will measure both the content and expression of several CA isoforms in order to determine which isoform(s) display an altered pattern of expression in this condition. Third, we will overexpress one or more CA isoforms in cultured muscle cells, and then measure malonyl-CoA production, lipid oxidation and insulin stimulated glucose transport. Thus we will directly determine whether CA overexpression impairs lipid metabolism and induces insulin resistance. These studies should provide important new data, therefore, on the CA family of enzymes, and their role in the insulin resistance.